A ligand-dependent signaling function exerted by the ErbB-3 protein has previously been found activated in human neoplasia. Employing a chimeric epidermal growth factor receptor (EGFR)/ErbB-3 receptor transfectant. it was shown that this function entails activation of cytoplasmic signaling pathways distinct from other family members. In particular. ErbB-3- mediated mitogenic signaling involved efficient recruitment of PI 3- kinase activity. while it lacked significant activation of phospholipase C (PLC)-gamma or GTPase activating protein (GAP). In search of a natural ligand. conditioned media and purified proteins were tested for induction of ErbB-3-specific tyrosine phosphorylation in mammary epithelial cells and NIH/3T3 transfectants expressing the ErbB-3 protein at high levels. Using this approach, it was shown that the bacterially expressed EGF domain of heregulin was able to trigger ErbB-3 tyrosine phosphorylation in vivo as well as of a related receptor. ErbB-4. when expressed at high levels in NIH/3T3 fibroblasts. Moreover. purification and microsequencing of a heparin-binding protein exhibiting ErbB-3 triggering activity has established that this protein was distinct from members of the heregulin family. Using degenerate oligonucleotides, specific DNA probes were generated by a polymerase chain reaction (PCR)-based approach and cDNA clones were isolated. The complete structural and functional characterization of this putative ErbB-3 ligand is pending. Finally. evidence was established that biological cooperation of several ErbB family members including ErbB-2 and ErbB-3 in a model system is paralleled in human tumors.